Asymmetric Transfer Hydrogenation as a Key Step in the Synthesis of the Phosphonic Acid Analogs of Aminocarboxylic Acids.

α-Aminophosphonic acids have a remarkably broad bioactivity spectrum. They can function as highly efficient transition state mimics for a variety of hydrolytic and angiotensin-converting enzymes, which makes them interesting target structures for synthetic chemists. In particular, the phosphonic acid analogs to α-aminocarboxylic acids (Pa AAs) are potent enzyme inhibitors, but many of them are only available by chiral or enzymatic resolution; sometimes only one enantiomer is accessible, and several have never been prepared in enantiopure form at all. Today, a variety of methods to access enantiopure α-aminophosphonic acids is known but none of the reported approaches can be generally applied for the synthesis of Pa AAs. Here we show that the phosphonic acid analogs of many (proteinogenic) α-amino acids become accessible by the catalytic, stereoselective asymmetric transfer hydrogenation (ATH) of α-oxo-phosphonates. The highly enantioenriched (enantiomeric excess (ee) ≥ 98 %) α-hydroxyphosphonates obtained are important pharmaceutical building blocks in themselves and could be easily converted to α-aminophosphonic acids in most studied cases. Even stereoselectively deuterated analogs became easily accessible from the same α-oxo-phosphonates using deuterated formic acid (DCO2 H).

Comprehensive reversed-phase×chiral two-dimensional liquid chromatography coupled to quadrupole-time-of-flight tandem mass spectrometry with post-first dimension flow splitting for untargeted enantioselective amino acid analysis.

This work describes a comprehensive achiral × chiral two-dimensional liquid chromatography separation for enantioselective amino acid analysis coupled to electrospray ionization-tandem mass spectrometry detection using data-independent acquisition. Flow splitting after the first and second dimension separation was utilized for volumetric flow reduction and for enabling a multi-detector approach (with ultraviolet, fluorescence, charged aerosol, and MS detection), respectively. Derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate provided a chromophore, a fluorophore, and an efficient mass tag for efficient ionization in positive electrospray ionization-mass spectrometry. Chiral columns often have limitations in terms of their chemoselectivity, which may be a problem when complex sample mixtures with structurally related compounds need to be separated. It can be alleviated by a reversed-phase×chiral two-dimensional-liquid chromatography setup, in which the first dimension provides the chemoselectivity and a chiral tandem column constituted of quinine-carbamate derived weak anion-exchanger and zwitterionic ion-exchanger in the second dimension separation of D- and L-amino acid enantiomers. The method was used to control the stereointegrity of the therapeutic peptide octreotide. After hydrolysis, all amino acid constituents were detected with the correct configuration and composition. Some options for flow splitting and integration of destructive detectors in the first dimension separation are outlined.

Comprehensive Online Reversed-Phase × Chiral Two-Dimensional Liquid Chromatography-Mass Spectrometry with Data-Independent Sequential Window Acquisition of All Theoretical Fragment-Ion Spectra-Acquisition for Untargeted Enantioselective Amino Acid Analysis.

This work presents an advanced analytical platform for untargeted enantioselective amino acid analysis (eAAA) by comprehensive achiral × chiral 2D-LC hyphenated to ESI-QTOF-MS/MS utilizing data-independent SWATH (sequential window acquisition of all theoretical fragment-ion spectra) technology. The methodology involves N-terminal pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) as retention, selectivity, and MS tag, supporting retention and UV detection in RPLC (1D), chiral recognition, and thus enantioselectivity by the core-shell tandem column composed of a quinine carbamate weak anion exchanger (QN-AX) and a zwitterionic chiral ion-exchanger (ZWIX(+)) (2D) as well as the ionization efficiency during positive electrospray ionization due to a high proton affinity of the AQC label. Furthermore, the urea-type MS tag gives rise to the generation of AQC-tag characteristic signature fragments in MS2. The latter allows the chemoselective mass spectrometric filtering of targeted and untargeted N-derivatized amino acids or related labeled species. The chiral core-shell tandem column provides a complete enantioselective amino acid profile of all proteinogenic amino acids within 1 min, with full baseline separation of all enantiomers, but without resolution of isomeric Ile/allo-Ile (aIle)/Leu, which can be resolved by RPLC. The entire LC × LC separation occurs within a total run time of 60 min (1D), with the chiral 2D operated in gradient elution mode and a cycle time of 60 s. A strategy to mine the 2D-LC-SWATH data is presented and demonstrated for the qualitative eAAA of two peptide hydrolysate samples of therapeutic peptides containing common and uncommon as well as primary and secondary amino acids. Absolute configuration assignment of amino acids using template matching for all proteinogenic amino acids was made feasible due to method robustness and the inclusion of an isotopically labeled L-[U-13C15N]-AA standard. The quantification performance of this LC × LC-MS/MS assay was also evaluated. Accuracies were acceptable for the majority of AAs enabling AA composition determination in peptide hydrolysates simultaneously with configuration assignment, as exemplified by oxytocin. This methodology represents a step toward truly untargeted 2D enantioselective amino acid analysis and metabolomics.

Discovery of Thanafactin A, a Linear, Proline-Containing Octalipopeptide from Pseudomonas sp. SH-C52, Motivated by Genome Mining.

Genome mining of the bacterial strains Pseudomonas sp. SH-C52 and Pseudomonas fluorescens DSM 11579 showed that both strains contained a highly similar gene cluster encoding an octamodular nonribosomal peptide synthetase (NRPS) system which was not associated with a known secondary metabolite. Insertional mutagenesis of an NRPS component followed by comparative profiling led to the discovery of the corresponding novel linear octalipopeptide thanafactin A, which was subsequently isolated and its structure determined by two-dimensional NMR and further spectroscopic and chromatographic methods. In bioassays, thanafactin A exhibited weak protease inhibitory activity and was found to modulate swarming motility in a strain-specific manner.

A population-based resource for intergenerational metabolomics analyses in pregnant women and their children: the Generation R Study.

INTRODUCTION: Adverse exposures in early life may predispose children to cardio-metabolic disease in later life. Metabolomics may serve as a valuable tool to disentangle the metabolic adaptations and mechanisms that potentially underlie these associations. OBJECTIVES: To describe the acquisition, processing and structure of the metabolomics data available in a population-based prospective cohort from early pregnancy onwards and to examine the relationships between metabolite profiles of pregnant women and their children at birth and in childhood. METHODS: In a subset of 994 mothers-child pairs from a prospective population-based cohort study among pregnant women and their children from Rotterdam, the Netherlands, we used LC-MS/MS to determine concentrations of amino acids, non-esterified fatty acids, phospholipids and carnitines in blood serum collected in early pregnancy, at birth (cord blood), and at child's age 10 years. RESULTS: Concentrations of diacyl-phosphatidylcholines, acyl-alkyl-phosphatidylcholines, alkyl-lysophosphatidylcholines and sphingomyelines were the highest in early pregnancy, concentrations of amino acids and non-esterified fatty acids were the highest at birth and concentrations of alkyl-lysophosphatidylcholines, free carnitine and acyl-carnitines were the highest at age 10 years. Correlations of individual metabolites between pregnant women and their children at birth and at the age of 10 years were low (range between r = - 0.10 and r = 0.35). CONCLUSION: Our results suggest that unique metabolic profiles are present among pregnant women, newborns and school aged children, with limited intergenerational correlations between metabolite profiles. These data will form a valuable resource to address the early metabolic origins of cardio-metabolic disease.

Derivatize, Racemize, and Analyze-an Easy and Simple Procedure for Chiral Amino Acid Standard Preparation for Enantioselective Metabolomics.

A simple, controllable, and reproducible stereoisomerization (racemization and epimerization) protocol for the preparation of scalemic α-amino acid mixtures from stereoisomerically pure standards was developed. Simply derivatize your amino acids with a racemization tag that incorporates a urea bond on the N-terminus of the target amino acid and incubate at elevated temperatures up to 95 °C for defined time periods until the targeted d-amino acid levels are obtained. The racemization tags investigated were 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC), aminophenyl- N-hydroxysuccinimidyl carbamate (AC), and 3-aminopyridyl- N-hydroxysuccinimidyl carbamate (APC). Employing this method, it was possible to create a ready-to-use, tailor-made chiral uniformly 13C and 15N labeled [U -13C15N]-amino acid standard with the desired d-amino acid percentage within minutes or hours without sample cleanup. A racemization time of 30 min at 95 °C will lead to a d-amino acid level of 1-5%, while 6 h at 95 °C provides 15-30% d-amino acids. Racemization occurs due to imine formation at the chiral carbon atom bound to the urea-linking group without decomposition of labile amino acids such as Asn, Gln, Trp, Cit, and theanine. For amino acids possessing two chiral centers such as dl-Ile or dl-Thr, only the epimerization of isomers with different stereochemistry at the second chiral center will produce all four possible isobaric enantiomers. All measurements were performed on the zwitterionic Chiralpak ZWIX(+) column using a dual hydro-organic flow gradient combined with HPLC-ESI-QTOF-MS analysis. This new racemization method solves the problem of (enantioselective) matrix effects and inaccurate results in LC-MS based enantioselective metabolomics and warrants full MS-compatibility.

A Methylidene Group in the Phosphonic Acid Analogue of Phenylalanine Reverses the Enantiopreference of Binding to Phenylalanine Ammonia-Lyases.

Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. MIO enzymes catalyze the stereoselective synthesis of α- or ß-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognizes promising herbicide candidates as well. The present study found that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the ß-position inhibited phenylalanine ammonia-lyases (PAL), representing MIO enzymes. X-ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene-substituted substrate analogue as being a mirror image relation to the natural l-phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)- and (S)-enantiomers is possible by a mirror image packing.

Apparent Saturation of Branched-Chain Amino Acid Catabolism After High Dietary Milk Protein Intake in Healthy Adults.

CONTEXT: Milk protein contains high concentrations of branched-chain amino acids (BCAA) that play a critical role in anabolism and are implicated in the onset of obesity and chronic disease. Characterizing BCAA catabolism in the postprandial phase could elucidate the impact of protein intake on obesity risk established in the "early protein hypothesis." OBJECTIVE: To examine the acute effects of protein content of young child formulas as test meals on BCAA catabolism, observing postprandial plasma concentrations of BCAA in relation to their degradation products. METHODS: The TOMI Add-On Study is a randomized, double-blind crossover study in which 27 healthy adults consumed 2 isocaloric young child formulas with alternating higher (HP) and lower (LP) protein and fat content as test meals during separate interventions, while 9 blood samples were obtained over 5 hours. BCAA, branched-chain α-keto acids (BCKA), and acylcarnitines were analyzed using a fully targeted HPLC-ESI-MS/MS approach. RESULTS: Mean concentrations of BCAA, BCKA, and acylcarnitines were significantly higher after HP than LP over the 5 postprandial hours, except for the BCKA α-ketoisovalerate (KIVA). The latter metabolite showed higher postprandial concentrations after LP. With increasing mean concentrations of BCAA, concentrations of corresponding BCKA, acylcarnitines, and urea increased until a breakpoint was reached, after which concentrations of degradation products decreased (for all metabolites except valine and KIVA and Carn C4:0-iso). CONCLUSION: BCAA catabolism is markedly influenced by protein content of the test meal. We present novel evidence for the apparent saturation of the BCAA degradation pathway in the acute postprandial phase up to 5 hours after consumption.

Endothelial epoxyeicosatrienoic acid release is intact in aldosterone excess.

BACKGROUND AND AIMS: Endothelial dysfunction (ED) is considered to be a major driver of the increased incidence of cardiovascular disease in primary aldosteronism (PA). The functionality of the epoxyeicosatrienoic acid (EET) pathway, involving the release of beneficial endothelium-derived lipid mediators, in PA is unknown. Evidence suggests this pathway to be disturbed in various models of experimental hypertension. We therefore assessed EET production in primary human coronary artery endothelial cells exposed to aldosterone excess and measured circulating EET in patients with PA. METHODS: We used qPCR to investigate changes in the expression levels of essential genes for the synthesis and degradation of EET, calcium imaging to address the functional impact on overall endothelial function, as well as mass spectrometry to determine endothelial synthetic capacity to release EET upon stimulation. RNA-seq was performed to gain further mechanistic insights. Eicosanoid concentrations in patient's plasma were also determined by mass spectrometry. RESULTS: Aldosterone, while eliciting proinflammatory VCAM1 expression and disturbed calcium response to acetylcholine, did not negatively affect stimulated release of endothelial EET. Likewise, no differences were observed in eicosanoid concentrations in plasma from patients with PA when compared to essential hypertensive controls. However, an inhibitor of soluble epoxide hydrolase abrogated aldosterone-mediated VCAM1 induction and led to a normalized endothelial calcium response probably by restoring expression of CHRNE. CONCLUSION: EET release appears intact despite aldosterone excess. Epoxide hydrolase inhibition may revert aldosterone-induced functional changes in endothelial cells. These findings indicate a potential new therapeutic principle to address ED, which should be explored in future preclinical and clinical trials.

Enantioseparation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids and other zwitterionic compounds on cinchona-based chiral stationary phases.

The fluorescent tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ Fluor reagent kit from Waters) is a commercial N-terminal label for proteinogenic amino acids (AAs), designed for reversed-phase separation and quantification of the AA racemates. The applicability of AQC-tagged AAs and AA-type zwitterionic compounds was tested for enantiomer separation on the tert-butyl carbamate modified quinine and quinidine based chiral stationary phases, QN-AX and QD-AX employing polar-organic elution conditions. The investigated test analytes included the enantiomers of the positional isomers of isoleucine (Ile), threonine, homoserine, and 4-hydroxyproline. Furthermore, ß-AAs, cyclic, and heterocyclic AAs including trans-2-amino-cyclohexane carboxylic acid and trans-2-aminocyclohexyl sulfonic acid, phenylalanine derivatives substituted with halides with increasing electronegativity and 3,4-dihydroxyphenylalanine, cysteine-related derivatives including homocysteic acid, methionine sulfone, cysteine-S-acetic acid, and cysteine-S-acetamide as well as a small range of aminophosphonic acids were enantioseparated. A mechanistic interaction study of AQC-AAs in comparison with fluoresceine isothiocyanate-labeled AAs was performed. The chiral and chemoselective recognition processes involved in enantiomer separation and retention was systematically discussed. Special emphasis was set on the influential factors exhibited by the chemistry, branching position, and spatial properties of the investigated zwitterionic analytes. The general interest to separate and distinguish between different types of branched-chained AAs and metabolic side products thereof lies in the toxicity of some of these compounds, which makes for instance allo-Ile an attractive candidate in disease-related biomarker research.

Automated sample preparation with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate and iodoacetamide derivatization reagents for enantioselective liquid chromatography tandem mass spectrometry amino acid analysis.

Enantioselective amino acid analysis is gaining increasing importance in pharmaceutical, biomedical and food sciences. While there are many methods available for enantiomer separation of amino acids, the simultaneous analysis of all chiral proteinogenic amino acids by a single method with one column and a single condition is still challenging. Herein, we report an enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS) assay using Chiralpak QN-AX as chiral column. With 6-aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) as derivatization reagent, efficient enantioselective separation of D- and L-amino acids using HPLC has become possible. Thiol-containing amino acids like Cys are alkylated prior to AQC-labelling. A protocol for automated sample preparation including both derivatization step and calibrator preparation is presented. For compensating matrix effects, u-13C15N-labelled internal standards (IS) were employed. The method was validated and applied to the enantioselective analysis of amino acids in a bacterial fermentation broth.

A prospective longitudinal cohort study on risk factors for COVID-19 vaccination failure (RisCoin): methods, procedures and characterization of the cohort.

The primary objective of the RisCoin study was to investigate the interplay of genetic, metabolic, and lifestyle factors as well as stress levels on influencing the humoral immune response after at least two COVID-19 vaccinations, primarily with mRNAs, and the risk of SARS-CoV-2 breakthrough infections during follow-up. Here, we describe the study design, procedures, and study population. RisCoin is a prospective, monocentric, longitudinal, observational cohort study. Between October and December 2021, 4515 participants with at least two COVID-19 vaccinations, primarily BNT162b2 and mRNA-1273, were enrolled at the LMU University Hospital of Munich, thereof > 4000 healthcare workers (HCW), 180 patients with inflammatory bowel disease under immunosuppression, and 119 patients with mental disorders. At enrollment, blood and saliva samples were collected to measure anti-SARS-CoV-2 antibodies, their neutralizing capacity against Omicron-BA.1, stress markers, metabolomics, and genetics. To ensure the confidential handling of sensitive data of study participants, we developed a data protection concept and a mobile application for two-way communication. The application allowed continuous data reporting, including breakthrough infections by the participants, despite irreversible anonymization. Up to 1500 participants attended follow-up visits every two to six months after enrollment. The study gathered comprehensive data and bio-samples of a large representative HCW cohort and two patient groups allowing analyses of complex interactions. Our data protection concept combined with the mobile application proves the feasibility of longitudinal assessment of anonymized participants. Our concept may serve as a blueprint for other studies handling sensitive data on HCW.

Performance evaluation of Mimetic Ligand™ B14-triazole-FractoAIMs adsorbents for the capture of human monoclonal immunoglobulin G from cell culture feed.

The new affinity-type Mimetic Ligand™ B14 was coupled with a 1,2-diaminoethane spacer (2LP) and a [1,2,3]-triazole spacer (TRZ) to three different support media. In addition to the agarose-based PuraBead and the polymethacrylate-type Fractogel, three new polymeric support media were introduced, the FractoAIMs 1, 2, and 3 (FA1, FA2, and FA3). These new FA supports differ in pore size as well as density of epoxide groups. The immobilization of the B14-ligand onto an azide-group-modified surface was performed with a copper (I)-mediated Click reaction. The IgG capture performance was tested for various ligand-spacer support combinations using cell culture feed containing human immunoglobulin G(1) (hIgG(1)). The most promising adsorbent, B14-TRZ-FA3, was further optimized by improving the surface chemistry through a triple endcapping concept employing an improved Click reaction protocol. This new technique enabled the most efficient deactivation of residual azide groups. In a direct comparison with a commercially available Protein A media, B14-TRZ-FA3 3× ec provided superior results at fast flow-rates and low bed-height. Dynamic binding capacities of 11.4 g/L for 10% breakthrough of hIgG(1), elution capacities of 16.0 g/L hIgG(1) and a recovery of 86% were achieved. The same results were obtained for a dialyzed and pre-purified feed solution, which is a clear indicator that triple-endcapped affinity support surfaces are practically inert to the non-specific binding of host cell proteins.

Enantioselective multiple heart cutting online two-dimensional liquid chromatography-mass spectrometry of all proteinogenic amino acids with second dimension chiral separations in one-minute time scales on a chiral tandem column.

In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient "smart peak parking" algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of <0.21% RSD were a requirement for the time-based sampling mode and finally led to a robust, fully automated enantioselective amino acid analysis platform. This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

Acute Metabolic Response in Adults to Toddler Milk Formulas with Alternating Higher and Lower Protein and Fat Contents, a Randomized Cross-Over Trial.

Protein intake in early life influences metabolism, weight gain, and later obesity risk. As such, a better understanding of the effects of protein intake on the postprandial metabolism and its dynamics over time may elucidate underlying mechanisms. In a randomized crossover study, we observed fasted adults who consumed two isocaloric toddler milk formulas concentrated as meals of 480 kcal with 67 g of carbohydrates 30 g (HP) or 7 g (LP) protein, and 10 g or 20 g fat, respectively. Anthropometry and body plethysmography were assessed, and blood samples collected at baseline and over five hours. Time-specific concentrations, areas under concentration curves (AUC), and maximum values of metabolites were compared by paired t-tests to examine the effects of protein content of toddler milks on postprandial plasma concentrations of insulin, glucose, branched-chain amino acids (BCAA), urea and triglycerides. Twenty-seven men and women aged 26.7 ± 5.0 years (BMI: 22.2 ± 2.5 kg/m2) (mean ± SD) participated. BCAA AUC, and Cmax values were significantly higher with HP than LP (144,765 ± 21,221 vs. 97,089 ± 14,650 µmol·min/L, p < 0.001; 656 ± 120 vs. 407 ± 66 µmol/L, p < 0.001), as were insulin AUC and Cmax values (6674 ± 3013 vs. 5600 ± 2423 µmol·min/L, p = 0.005; 71 ± 37 vs. 55 ± 28 µmol/L, p = 0.001). Higher glucose, urea, and triglyceride concentrations occurred in the late postprandial phase (≥180 min) with HP. In conclusion, we noted that higher milk protein intake induces increased postprandial BCAA concentrations for at least 5 h and led to higher initial insulin secretion. Gluconeogenesis due to an influx of amino acids and their degradation after HP meal might explain the late effects of protein intake on glucose and insulin.

Curing Cats with Feline Infectious Peritonitis with an Oral Multi-Component Drug Containing GS-441524.

Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn® in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn®. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn® displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn® containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species.

Multiple Micronutrients, Lutein, and Docosahexaenoic Acid Supplementation during Lactation: A Randomized Controlled Trial.

Breastfed infants require an adequate supply of critical nutrients for growth, tissue functions, and health. Recommended intakes for several nutrients are considerably higher in lactating than non-lactating women but are not always met with habitual diets. We report a randomized, double-blind clinical trial in 70 healthy lactating women in Germany evaluating the effects of supplementation with multiple micronutrients, lutein, and docosahexaenoic acid (DHA) compared to placebo on maternal nutrient status and milk composition. The primary endpoint was the effect on the change of human milk DHA content (as a proportion of total milk fatty acids) during 12 weeks of supplementation. Maternal blood and milk biomarkers were measured as secondary endpoints. Supplementation increased maternal milk DHA by 30% compared to a decline in the placebo group. Supplementation also increased maternal blood DHA (17%), eicosapentaenoic acid (4%), 25-OH-vitamin D (24%), vitamin B12 (12%), lutein (4%), and beta carotene (49%), while homocysteine decreased. No significant difference in the number of adverse events was observed between supplementation and placebo groups. In conclusion, multi-micronutrient supplementation was safe and increased maternal blood and milk concentrations of selected nutrients in healthy women.

Stereoselective separation of underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatized amino acids using zwitterionic quinine and quinidine type stationary phases by liquid chromatography-High resolution mass spectrometry.

Amino acids play an important role in cellular processes and are building blocks for peptides and proteins, which take part in regulatory processes within each organism. Hence a large variety of biotechnologically or synthetically produced therapeutic drugs are peptides and proteins. Due to the chiral nature of amino acids and the large variety of common, uncommon and newly synthesized amino acid type compounds, stereoselective separation tools combined with mass spectrometric detection are important in research as well as purity control of therapeutics in industry. Since structural isomers and epimers of common amino acids are isobaric to each other, stereoselective separation is key to their identification. For this purpose zwitterionic quinine and quinidine type chiral stationary phases Chiralpak ZWIX(+) and Chiralpak ZWIX(-) were investigated for their separation performance for underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) derivatized proteinogenic amino acids, uncommon amino acids and their isobaric analogs such as allo-threonine, homoserine, allo-isoleucine and homocysteine by HPLC-ESI-QTOF-MS. Cystine and homocystine were reduced with dithiothreitol and S-alkylated with iodoacetic acid and iodoacetamide. In general, derivatization with AQC and thiol alkylation increased the detection sensitivity and resolution of acidic, basic and polar amino acids significantly (e.g. separation factor of Asp increased from 1.00 to 2.29 for Asp-AQC). In addition, throughout this study a u-13C15N-L-amino acid metabolomics mixture was added to the DL-amino acid test solution and used as a co-eluting peak assignment standard to identify the corresponding u-12C14N-L-amino acid peak and hence determine the elution order of the enantiomer pairs for complex mixtures within a single run, employing the same separation conditions for underivatized and AQC-derivatized amino acids and their isobaric analogs.

Racemization without deamidation: Effect of racemization conditions on 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate tagged amino acids.

The aim of this research study was to provide a more thorough understanding of the underlying mechanism and to broaden the application field of the recently introduced racemization method employing the amino acid derivatization tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC, AccQ) for heat-induced stereoisomerization of common amino acids as well as uniformly isotopically labeled [U-13C15N]-amino acids. The influence of different buffer types such as sodium borate buffer and sodium carbonate buffer as well as their pH and molarity on the racemization and deamidation of amino acids were investigated. It was found that a 0.4 M borate buffer with a pH of 8.0 +/- 0.2 was the most suitable derivatization as well as racemization buffer to ensure degradation free racemization of deamidation prone compounds such as glutamine. Hereby essential was the in-solution pH measurement before and after derivatization with AQC as well as after heat-induced racemization. This strategy provided further insight at which pH an actual racemization event was observed and when an unwanted deamidation of glutamine to glutamic acid occurred. In addition also the influence of the presence of oxygen during racemization was studied. In this context it was possible to determine ideal oxidation and racemization conditions for the production of scalemic mixtures of chiral isotopically labeled methionine AQC-DL-[U-13C15N]-Met as well as its oxidation products, AQC-DL-[U-13C15N]-Met-O and AQC-DL-[U-13C15N]-Met-O2. All stereoselective separations were performed on the zwitterionic Chiralpak ZWIX(+) column combined with HPLC-ESI-QTOF-MS analysis in positive ionization mode.

Protein A- and Protein G-gold nanoparticle bioconjugates as nano-immunoaffinity platform for human IgG depletion in plasma and antibody extraction from cell culture supernatant.

Detection of disease-related biomarkers in plasma provides a possibility for early clinical diagnosis. However, highly abundant proteins in plasma, such as human immunoglobulin (hIgG) are a main impediment to biomarker discovery and analysis. Therefore, rapid and easy depletion of hIgG in the plasma is beneficial for biomarker discovery. In this work, citrate-capped gold nanoparticles (GNPs) were synthesized and conjugated with cysteine-tagged recombinant Protein A (rProtA) and Protein G (ProtG), respectively. The resultant protein-GNP bioconjugates were thoroughly characterized by surface plasmon resonance spectroscopy, hydrodynamic light scattering (DLS), electrophoretic light scattering (ELS) and rotary metal shadowing transmission electron microscopy (TEM) measurements. In order to quantitatively control the amount of the rProt A and ProtG on the GNP surface, binding studies and isotherm measurements have been performed. rProtA-GNP conjugate exhibited better binding capacities towards hIgG. Its surface coverage with rProtA molecules was determined by protein quantification after hydrolysis of the rProtA-GNP conjugate, GNP removal and subsequent amino acid assay by HPLC with fluorescence detection. Binding isotherms acquired with hIgG revealed their maximal capacity for depletion experiments. Depletion efficiency of around 90% could be achieved in a standard solution. With optimized amount of rProtA-GNP and ProtG-GNP, respectively, hIgG could be efficiently extracted from real samples (human plasma and hIgG-spiked cell culture supernatant). A benchmarking study with ProteinA-modified magnetic particles (Dynabeads) was performed as well. The results document that these rProtA-GNP and ProtG-GNP affinity nanoparticles could be a promising alternative to magnetic bead based immunoaffinity trapping and constitutes a flexible platform for both depletion of hIgG from human plasma and antibody affinity capture from cell culture supernatants in process control of biopharmaceuticals by simple solution handling (via pipetting) and centrifugation steps.